Over 350 million people worldwide are chronically infected carriers of hepatitis B (HBV). HBV is a virus that infects the liver and causes an increased risk of chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma (cancer of the liver). Hepatitis B is the cause of over 80 percent of hepatocellular carcinomas, and claims the lives of 1-2 million people worldwide every year, representing an important public health challenge and a growing market for new therapeutics.
The severity of hepatitis B infection depends on the state of the infected person's immune system at the time of infection. Hepatitis B is most debilitating when it is transmitted from a mother to her baby at birth, as the immune system of an infant is typically not capable of mounting an effective response against the virus. As a result, chronic infection occurs in 90 percent of infants that are infected at birth, and the risk of hepatocellular carcinoma is much higher (20 percent to 30 percent). If infection occurs at 1-5 years of age, the risk of chronic infection drops to 25-50 percent. If infection occurs in late childhood or adulthood, the chances of chronic infection are only 2-6 percent. Hepatocellular carcinoma rarely occurs in people who become chronically infected as adults.
Chronic carriers are highly infectious, and fall into two general categories: (1) asymptomatic chronic persistent hepatitis B, where most chronic carriers do not seek medical attention for their condition, and (2) chronic active hepatitis B that is more serious, but less common, than chronic persistent hepatitis. When symptoms are present in patients with asymptomatic chronic persistent HBV, those symptoms may be relatively minor such as fatigue, abdominal pains, weakness, fever, and intolerance to fat or alcohol. The disease does not usually progress to severe liver disease, but a few patients may develop chronic active hepatitis B. The consequences of chronic active hepatitis B include cirrhosis of the liver and primary hepatocellular carcinoma (PHC). In cirrhosis of the liver, fibrous tissue forms, replacing damaged liver cells. The liver then becomes hardened, enlarged and distorted, and may eventually fail. PHC is relatively rare in areas of low hepatitis B endemicity but is very common, and a frequent cause of death, in areas of high endemicity.
Current treatment for chronic hepatitis B involves taking injections of interferon alfa-2, for four months. There are four brands of interferon alfa-2 approved in the United States: Schering Plough's Intron® A, Amgen's Infergen, Hoffmann-La Roche's Roferon, and GlaxoSmithKline's Wellferon. Intron® A is the only form of alpha interferon that is approved for hepatitis B, the others are approved for hepatitis C only.
Interferon alpha is believed to increase the number of MHC Class I molecules on the surface of liver cells, thereby increasing the ability of immune cells to recognize and destroy the infected liver cells. Interferon alpha also increases the amount of ribonuclease enzymes that cleave HBV-RNA in liver cells, impeding HBV growth.
Although interferon alpha can completely eliminate chronic hepatitis B infections in some people, its use is limited because over half of all patients do not respond to treatment. In people with chronic hepatitis B, interferon alpha may slow the disease by reducing the amount of virus in their bodies and slowing the damage to their livers.
The side effects of interferon alpha-2 treatment can be so debilitating that patients are recommended to take a week or two off work when beginning treatment. The most common side effect are symptoms of the flu-fatigue, fever, muscle pains, general body aches, chills, and nausea. Mild hair thinning and dry, itchy skin can also occur.
Antiviral agents and therapeutic vaccines are being investigated as possible alternative treatment options due to the ineffectiveness and side effects of interferon alpha therapy. Promising results have been seen with second generation nucleoside analogues, such as lamivudine and famciclovir. Zeffix® (lamivudine), a nucleoside reverse transcriptase inhibitor, is a promising single drug candidate as a treatment for chronic hepatitis B, and received FDA approval to be marketed and sold in the United States in 1999. Other antiviral agents under evaluation include BMS200, 475, ganciclovir, and adefovir dipivoxil. Combination therapy of the above candidates with interferon alpha is also being investigated.
Hoffman La Roche and Schering Plough Corporation have recently applied to the U.S. Food and Drug Administration (FDA) for marketing approval of their versions of so-called pegylated interferons named PEGASYS™ (Hoffman La Roche) and PEG-INTRON™ (Schering Plough Corp.). Pegylated interferon are alpha interferons that are modified by polyethylene glycol (PEG) so that they can be given once a week and provide a sustained level of interferon within the patient. The pegylated formulations may avoid the peaks and troughs of interferon levels and interferon side effects that occur when given three times a week. Pegylated interferons may be especially beneficial to those who have relapsed following monotherapy or combination therapy.
Vaccine approaches have been attempted to treat chronic hepatitis. Couillin and colleagues [Couillin et al. (1999) J. of Infect. Dis., 180: 15-26] evaluated whether vaccination with hepatitis B surface antigen (HBsAg) was able to overcome the tolerance to HBsAg in patients with chronic hepatitis. They determined that HBsAg was effective in a fraction of the population.
Studies have also been performed in animal models to evaluate whether an immune response can be induced in animal models of the disease. Thus, Bocher and colleagues [Bocher et al. (2001) E. J. of Immun., 31:2071-2079] evaluated the immune response towards vaccination in a humanized (trimera) mouse model. As a model of the disease, these authors transferred PBMCs from patients chronically infected with hepatitis B into the mice, and then vaccinated the mice with hepatitis B core protein (HBc) or DNA coding for hepatitis B core protein. The authors noted that HBc-specific T-helper-cell and B-cell responses were induced when the mice were immunized with HBc or with DNA coding for HBC. The authors noted that either HBc protein or HBc-encoding DNA could represent candidate vaccines for therapeutic vaccination against chronic hepatitis B infection. It should be noted that in these studies very large doses of HBc were required to induce an immune response. The immune response in mice grafted with PBMCs from infected individuals could further be enhanced by the addition of immunostimulatory oligonucleotides (ISN).
In addition to considering active vaccination, passive transfer of immunity has been attempted: Lau and colleagues [Lau et al. (2002) Gastroenterology, 122:614-624] demonstrated that bone-marrow transfers from HBV-immune individuals to chronically infected individuals resulted in resolution of the infection. The resolution was associated with the transfer of T-cells reactive to HBc, leading those authors to postulate that therapeutic immunization with HBc protein or [HBc-encoding] DNA deserves investigation in patients with chronic hepatitis B infection.
Hepatitis B core protein (HBc) has therefore been recognized as a potentially useful antigen for therapeutic vaccination against chronic hepatitis B infection. Several problems however, have to be overcome to turn that potential into practice: the recombinant production of HBc is difficult. As discussed hereinafter the yield of production is very low, possibly because of the inherent nucleic-acid binding property of the HBc protein, and the resulting virus-like-particle (VLP) is furthermore difficult to purify to a level acceptable to regulatory authorities.
As a result of the difficulties associated with manufacturing HBc, alternative approaches have been pursued to induce an immune response to HBc in individuals chronically infected with HBV. Thus, for example, WO 01/16163 assigned to Hultgren and Sallberg proposes the use of multiple overlapping synthetic peptides comprising several amino acid residue sequences spanning the position 1-183 sequence of HBc. These inventors suggested that immunization with a mixture of peptides spanning the entire protein may induce an immune response that promotes clearance of the virus in chronically infected individuals. DNA encoding the HBc protein has been used to immunize chimpanzees chronically infected with HBV [Sallberg et al., (1998) Human Gene Therapy 10:1719-1729]. The use of DNA encoding a protein overcomes the requirement for purification of the protein, but DNA-vaccination has not been associated with a significant rate of success in humans.
U.S. Pat. No. 6,020,167 to Thoma discloses a vaccine that is said to be useful in treating chronic HBV infection. This vaccine comprises a polypeptide having one or more HBV pre-S1 or HBV core T-cell activating epitopes bound to a carrier capable of presenting the polypeptide. Particle-forming carriers were said to be preferred, with complete or substantial parts of the HBV core and surface proteins (HBc and HbsAg, respectively) being claimed carriers. As is discussed hereinafter, the complete core protein tends to bind nucleic acids, which can be problematic for vaccine manufacture. In addition, core molecules that are carboxy-terminally truncated to alleviate the nucleic acid binding, may be unstable and can provide a heterogeneous mixture in a vaccine.
The family hepadnaviridae are enveloped DNA-containing animal viruses that can cause hepatitis B in humans (HBV). The hepadnavirus family includes hepatitis B viruses of other mammals, e.g., woodchuck (WHV), and ground squirrel (GSHV), and avian viruses found in ducks (DHV) and herons (HeHV). Hepatitis B virus (HBV) used herein refers to a member of the family hepadnaviridae that infects mammals, as compared to a virus that infects an avian host, unless the discussion refers to a specific example of a non-mammalian virus.
The nucleocapsid or core of the mammalian hepatitis B virus (HBV or hepadnavirus) contains a sequence of 183 or 185 amino acid residues, depending on viral subtype, whereas the duck virus capsid contains 262 amino acid residues. Hepatitis B core protein monomers of the several hepadnaviridae self-assemble in infected cells into stable aggregates known as hepatitis B core protein particles (HBc particles). Two three-dimensional structures are reported for HBc particles. A first that comprises a minor population contains 90 copies of the HBc subunit protein as dimers or 180 individual monomeric proteins, and a second, major population that contains 120 copies of the HBc subunit protein as dimers or 240 individual monomeric proteins. These particles are referred to as T=4 or T=3 particles, respectively, wherein “T” is the triangulation number. These HBc particles of the human-infecting virus (human virus) are about are about 30 or 34 nm in diameter, respectively. Pumpens et al. (1995) Intervirology, 38:63-74; and Metzger et al. (1998) J. Gen. Viol., 79:587-590.
Conway et al., (1997) Nature, 386:91-94, describe the structure of human HBc particles at 9 Angstrom resolution, as determined from cryo-electron micrographs. Bottcher et al. (1997), Nature, 386:88-91, describe the polypeptide folding for the human HBc monomers, and provide an approximate numbering scheme for the amino acid residues at which alpha-helical regions and their linking loop regions form. Zheng et al. (1992), J. Biol. Chem., 267(13):9422-9429 report that core particle formation is not dependent upon the arginine-rich C-terminal domain, the binding of nucleic acids or the formation of disulfide bonds based on their study of mutant proteins lacking one or more cysteines and others' work with C-terminal-truncated proteins [Birnbaum et al., (1990) J. Virol. 64, 3319-3330].
The hepatitis B nucleocapsid or viral core protein (HBc) has been disclosed as an immunogenic carrier moiety that stimulates the T cell response of an immunized host animal. See, for example, U.S. Pat. No. 4,818,527, No. 4,882,145 and No. 5,143,726. A particularly useful application of this carrier is its ability to present foreign or heterologous B cell epitopes at the site of the immunodominant loop that is present at about residue positions 70-90, and more usually recited as about positions 75 to 85 from the amino-terminus (N-terminus) of the protein. Clarke et al. (1991) F. Brown et al. eds., Vaccines 91, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp.313-318.
During viral replication, HBV nucleocapsids associate with the viral RNA pre-genome, the viral reverse transcriptase (Pol), and the terminal protein (derived from Pol) to form replication competent cores. The association between the nucleocapsid and the viral RNA pre-genome is mediated by an arginine-rich domain at the carboxyl-terminus (C-terminus). When expressed in heterologous expression systems, such as E. coli where viral RNA pre-genome is absent, the protamine-like C-terminus; i.e., residues at positions 150 through 183, can bind E. coli RNA. Zhang et al. (1992) JBC, 267(13) 9422-29.
In an application as a vaccine moiety, it is preferable that the HBV nucleocapsids not bind nucleic acid derived from the host. Birnbaum et al. (1990) J. Virol., 64:3319-3330 showed that the protamine-like C-terminal domain of HBV nucleocapsids could be deleted without interfering with the protein's ability to assemble into virus-like particles. It is thus reported that proteins truncated to about position 144; i.e., containing the HBc sequence from position one through about 144, can self-assemble, whereas deletions beyond residue 139 abrogate capsid assembly [Birnbaum et al., (1990) J. Virl., 64: 3319-30; and Seifer et al., (1995) Intervirology, 38:47-62].
Zlotnick et al., (1997) Proc. Natl. Acad. Sci., USA, 94:9556-9561 studied the assembly of full length and truncated HBc proteins in to particles. In addition to discussing full length molecules, those authors reported the preparation of a truncated protein that contained the HBc sequence from position 1 through 149 in which the cysteines at positions 48, 61 and 107 were each replaced by alanines and in which a cysteine residue was added at the C-terminus (position 150). That C-terminal mercaptan was used for linkage to a gold atom cluster for labeling in electron microscopy.
More recently, Metzger et al. (1998) J. Gen. Viol., 79:587-590 reported that the proline at position 138 (Pro-138 or P138) of the human viral sequence is required for particle formation. Those authors also reported that assembly capability of particles truncated at the carboxy-terminus to lengths of 142 and 140 residues was affected, with assembly capability being completely lost with truncations resulting in lengths of 139 and 137 residues.
Several groups have shown that truncated particles exhibit reduced stability relative to standard hepatitis B core particles [Galena et al. (1989) J. Virol., 63:4645-4652; Inada, et al. (1989) Virus Res., 14:27-48], evident by variability in particle sizes and the presence of particle fragments in purified preparations [Maassen et al., (1994) Arch. Virol., 135:131-142). Thus, prior to the report of Metzger et al., above, Pumpens et al., (1995) Intervirology, 38:63-74 summarized the literature reports by stating that the carboxy-terminal border for HBc sequences required for self-assembly was located between amino acid residues 139 and 144, and that the first two or three amino-terminal residues could be replaced by other sequences, but elimination of four or eleven amino-terminal residues resulted in the complete disappearance of chimeric protein in transformed E. coli cells.
Recombinantly-produced hybrid HBc particles bearing internal insertions (referred to in the art as HBc chimeric particles or HBc chimers) containing various inserted polypeptide sequences have been prepared by heterologous expression in a wide variety of organisms, including E. coli, B. subtilis, Vaccinia, Salmonella typhimurium, Saccharomyces cerevisiae. See, for example Pumpens et al. (1995) Intervirology, 38:63-74, and the citations therein that note the work of several research groups.
Such HBc chimers often appear to have a less ordered structure, when analyzed by electron microscopy, compared to particles that lack heterologous epitopes [Schodel et al., (1994) J. Exp. Med., 180:1037-1046]. In some cases the insertion of heterologous epitopes into C-terminally truncated HBc particles has such a dramatic destabilizing affect that hybrid particles cannot be recovered following heterologous expression [Schodel et al. (1994) Infect. Immunol., 62:1669-1676]. Thus, many chimeric HBc particles are so unstable that they fall apart during purification to such an extent that they are unrecoverable or they show very poor stability characteristics, making them problematic for vaccine development.
The above Pumpens et al. (1995) Intervirology, 38:63-74 report lists particle-forming chimers in which the inserted polypeptide sequence is at the N-terminus, the C-terminus and between the termini. Insert lengths reported in that article are 24 to 50 residues at the N-terminus, 7 to 43 residues internally, and 11 to 741 residues at the C-terminus.
Kratz et al., (1999) Proc. Natl. Acad. Sci., U.S.A., 96:1915-1920 recently described the E. coli expression of chimeric HBc particles comprised of a truncated HBc sequence internally fused to the 238-residue green fluorescent protein (GFP). This chimer contained the inserted GFP sequence flanked by a pair of glycine-rich flexible linker arms replacing amino acid residues 79 and 80 of HBc. Those particles were said to effectively elicit antibodies against native GFP in rabbits as host animals.
U.S. Pat. No. 5,990,085 describes two fusion proteins formed from an antigenic bovine inhibin peptide fused into (i) the immunogenic loop between residues 78 and 79 and (ii) after residue 144 of carboxy-terminal truncated HBc. Expressed fusion proteins were said to induce the production of anti-inhibin antibodies when administered in a host animal. The titers thirty days after immunization reported in that patent are relatively low, being 1:3000-15,000 for the fusion protein with the loop insertion and 1:100-125 for the insertion after residue 144.
U.S. Pat. No. 6,231,864 teaches the preparation and use of a strategically modified hepatitis B core protein that is linked to a hapten. The modified core protein contains an insert of one to about 40 residues in length that contains a chemically reactive amino acid residue to which the hapten is pendently linked.
WO 01/27281 teaches that the immune response to HBc can be changed from a Th1 response to a Th2 response by the presence or absence, respectively, of the C-terminal cysteine-containing sequence of the native molecule. That disclosure also opines that disulfide formation by C-terminal cysteines could help to stabilize the particles. The presence of several residues the native HBc sequence immediately upstream of the C-terminal cysteine was said to be preferred, but not required. One such alternative that might be used to replace a truncated C-terminal HBc sequence was said to include a C-terminal cysteine and an optional sequence that defines an epitope from other than HBc.
Published PCT application WO 01/98333 teaches the deletion of one or more of the four arginine repeats present at the C-terminus of native HBc, while maintaining the C-terminal cysteine residue. That application also teaches that the deleted region can be replaced by an epitope from a protein other than HBc so that the HBc portion of the molecule so formed acts as a carrier for the added epitope.
Published PCT applications corresponding to PCT/US01/25625 (WO 02/13765 A2 published Feb. 21, 2002) and PCT/US01/41759 (WO 02/14478 A2 published Feb. 21, 2002) teach that stabilization of C-terminally truncated HBc particles can be achieved through the use of one or more added cysteine residues in the chimer proteins from which the particles are assembled. Those added cysteine residues are taught to be at on near the C-terminus of the chimeric protein.
A structural feature whereby the stability of full-length HBc particles could be retained, while abrogating the nucleic acid binding ability of full-length HBc particles, would be highly beneficial in vaccine development using the hepadnaviral nucleocapsid delivery system. Indeed, Ulrich et al. in their recent review of the use of HBc chimers as carriers for foreign epitopes [Adv. Virus Res., 50: 141-182 (1998) Academic Press] note three potential problems to be solved for use of those chimers in human vaccines. A first potential problem is the inadvertent transfer of nucleic acids in a chimer vaccine to an immunized host. A second potential problem is interference from preexisting immunity to HBc. A third possible problem relates to the requirement of reproducible preparation of intact chimer particles that can also withstand long-term storage.
The above four published PCT applications appear to contain teachings that can be used to overcome the potential problems disclosed by Ulrich et al. As disclosed hereinafter, the present invention provides another HBc chimer that provides unexpectedly high titers of antibodies against influenza, and in one aspect also provides a solution to the problems of HBc chimer stability as well as the substantial absence of nucleic acid binding ability of the construct. In addition, a contemplated recombinant chimer exhibits minimal, if any, antigenicity toward preexisting anti-HBc antibodies.
The above particle instability findings related to N-terminal truncated HBc chimer molecules notwithstanding, Neirynck et al., (October 1999) Nature Med., 5(10):1157-1163 reported that particle formation occurred on E. coli expression of a HBc chimer that contained the N-terminal 24-residue portion of the influenza M2 protein fused at residue 5 to full length HBc.
The previously discussed use of hybrid HBc proteins with truncated C-termini for vaccine applications offers several advantages over their full-length counterparts, including enhanced expression levels and lack of bound E. coli RNA. However, C-terminally truncated particles engineered to display heterologous epitopes are often unstable, resulting in particles that either fail to associate into stable particulate structures following expression, or that readily dissociate into non-particulate structures during and/or following purification. Such a lack of stability is exhibited by particles comprised of chimeric HBc molecules that are C-terminally truncated to HBc position 149 and also contain the above residues 1-24 of the influenza A M2 protein.
Others have reported that in wild type hepadnaviral core antigens a cysteine residue upstream of the HBcAg start codon is directly involved in the prevention of particle formation [Schodel et al. (Jan. 15, 1993) J. Biol. Chem., 268(2):1332-1337; Wasenauer et al. (March 1993) J. Virol., 67(3):1315-1322; and Nassal et al. (July 1993) J. Virol., 67(7):4307-4315]. All three groups reported that in wild type HBeAg, the cysteine residue at position −7 of the pre-core sequence, which is present when the core gene is translated from an upstream initiator methionine at position −30, is responsible for preventing particle formation and therefore facilitating the transition from particulate HBcAg to secreted, non-particulate HBeAg.
One aspect of the present invention discussed hereinafter is to provide a protein immunogen intended for administration to individuals chronically infected with hepatitis B virus that overcomes the above-mentioned problems of production and contamination. Furthermore the protein has been engineered to maintain physical stability, and to induce an immune response particularly useful for clearing the body of an existing hepatitis B viral infection.
The present invention described in detail hereinafter provides a vaccine treatment for chronic hepatitis that overcomes several of the previously observed problems with vaccines. Thus, a contemplated vaccine induces an enhanced immune response by providing T cell activation that is particularly useful for clearing the body of an existing hepatitis B viral infection and utilizes a carrier molecule that is stable and homogeneous while also being substantially free from nucleic acid binding.